The present invention relates to a process for the preparation of phosphatidylserines.
More particularly, the invention relates to a process for the preparation of phosphatidylserines starting from natural or synthetic phospholipids, by reacting them with Serine in an aqueous medium.
Phosphatidylserines have manifold importance, in particular in the production of pharmaceutical compositions suitable for the therapy of cerebral involutive disorders of different nature, such as senile decline or vascular pathologies, in the preparation of particular liposome formulations, and, more recently, the commercialisation of dietetic compositions containing natural lecithins, in particular soy lecithin, enriched in phosphatidyl-(L)-Serine, hereinafter referred to as PS, also containing polyunsaturated fatty acid acyl residues.
As industrial amounts of low-cost PS are increasingly needed, applicants carried out an extensive study in order to find conditions for the preparation of this product fulfilling the requirements of practicability on an industrial scale.
U.S. Pat. No. 5,700,668, in, discloses a process for the preparation of PS, which, contrary to those of the prior art (see, in particular, Comfurius P et al., Biochim. Biophys. Acta 488, 36 (1977); Yamane et al., Biochim. Biophys. Acta 1003, 277 (1989); JP-A-63 036,791; JP-A-02 079,990 and J.Chem.Soc. Perkin Trans. 1, 919 (1995)) allows to prepare PS in good yields on an industrial scale also from unpurified starting materials and without need for chromatographic purifications.
The process disclosed in said Patent comprises the reaction of natural or synthetic phosphatides with Serine in a diphasic system consisting of a solution of the starting phosphatide in an organic solvent, typically toluene, and of an aqueous solution containing Serine and phospholipase D. Keeping said system thoroughly stirred, phosphatides are transformed into PS which can be recovered from the organic phase by precipitation with acetone.
A similar process, although providing phosphatidylserine on large scale in good yields, deviates from the present trend for industrial chemical processes involving reduced use of organic solvents and minimizing by-products. This process, in fact, involves the use (and the recycle) of remarkable amounts of organic solvents and their recovery both during the reaction and during the isolation of the end product.
The present invention aims at providing a process for the preparation of phosphatidylserines on an industrial scale, which overcomes the drawbacks mentioned above with reference to the prior art.
This problem has been solved, according to the invention, by a process for the preparation of phosphatidylserines of formula (I) 
in which R1 and R2 are independently saturated, monounsaturated or polyunsaturated acyl C10-C30,
X=OH or OM, wherein M=alkali or alkaline-earth metal, ammonium, alkylammonium (including the inner salt),
comprising the reaction of phosphatides of general formula (II) 
in which R1, R2 and X have the meanings defined above and R3=CH2xe2x80x94CH2xe2x80x94NH2 or CH2xe2x80x94CH2xe2x80x94N30 (CH3)3,
with racemic or enantiomerically pure Serine, preferably with (L)-Serine, in the presence of a phospholipase D (PLD), characterized in that the reaction is carried out in an aqueous dispersion preferably in the presence of calcium salts. Serine is usually added to a buffered aqueous dispersions of phosphatides.
The starting material used in the process can be either a synthetic phosphatidylcholine (PC) such as DiMyristoylPhosphatidylCholine (DMPC), or a natural phosphatidylcholine or a mixture of phospholipids such as soy or egg lecithin or one of the commercially available phospholipid mixtures obtained by partial purification of soy or egg lecithin.
As far as the cost of the process is concerned, it is particularly convenient to use starting materials derived from lecithin in view of both their lower cost and the fact that phosphatidylethanolamine (PE), which is an important component of these mixtures, is transformed into PS under these reaction conditions.
The starting material should be thoroughly dispersed in an aqueous medium before the addition of the other reagents, in order to promote reactivity.
The dispersion is carried out keeping the starting phospholipide material under stirring for 1-10 hours at 20-50xc2x0 C. with 3-10 volumes of water or saline solutions.
The use of surfactants in this step promotes the dispersion of the substrate and therefore the reaction rate.
Ionic or non-ionic surfactants can be conveniently used, more conveniently the surfactant is bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) or Tween 80(R) in amounts from 0.01 to 0.4 g per gram of substrate.
The effect of these surfactants is particularly favourable when using phosphatidylcholines, such as DMPC, which have poor tendency to disperse in aqueous medium.
A further feature of this invention is that it is possible to easily remove the alcohol solvents, and in particular ethanol, which often contaminates the preparations of natural phospholipids (for example Phosphatidylcholine-enriched fractions obtained from soy lecithin) and which interfere in the subsequent reaction of formation of PS as they react in the presence of phospholipase D to give the corresponding phosphatidyl derivatives (such as phosphatidylethanol when ethanol is present in the mixture).
This removal can be conveniently carried out by dispersing the starting phospholipid in an aqueous saline solution, decanting the suspension and removing the liquid phase which contains ethanol dissolved therein.
As aqueous saline solution can be used any saline solution having sufficiently high density to easily separate the starting material, preferably a solution of calcium chloride or of Sodium Acetate/Acetic acid buffer or of both of them.
The starting material aqueous dispersion is added with an aqueous solution containing Serine, Calcium Chloride, a uffer to control pH and phospholipase D.
The amount of Serine will conveniently range from 4 to 20 mols of Serine per mol of phospholipid to be converted nto PS. Serine can be in the D, L or racemic forms to obtain in any case the corresponding phosphatidyl derivatives.
Calcium chloride is the source of calcium ion which favours the Phospholipase D-catalysed reactions; furthermore, the presence of calcium ion induces the separation of Phosphatidylserine in an easily filterable form after completion of the reaction.
The concentration of calcium chloride preferably ranges from 0.05 to 0.5 M.
The pH of the aqueous solution will depend on the origin of the used enzyme and it will preferably range from pH=4 to pH=9 included. In case of enzymes exerting their activity in substantially acid medium, the aqueous solution will be preferably buffered with 0.02M to 0.2M acetate buffer.
An enzyme having a transphosphatidylating activity higher than the hydrolysing activity should conveniently be used as the enzyme for carrying out the reaction; an enzyme of fermentative origin may be advantageously used, more conveniently the enzyme obtained by fermentation of the microorganism deposited at the ATCC under the number 55717.
The enzyme can be used either in the crude form, namely the fermentation mixture after filtering off the microorganism, or, more preferably, after purification by ultrafiltration through membrane with suitable cut-off to remove low molecular weight impurities. The enzyme-rich solution from the ultrafiltration can be used as it is or it can be freeze-dried to obtain the enzyme in the solid form; the enzyme amount preferably ranges from 10 to 100 units per gram of phosphatide (the enzymatic activity is determined with the test described in Biotechn Techn 7 795 (1993)).
The reaction is preferably carried out at 25-60xc2x0 C., most preferably from 40 to 50xc2x0 C.
PS is recovered by filtration of the reaction mixture, washing the solid with water to remove Serine and the present salts.
A particularly advantageous aspect of the process according to the invention is that the reaction product (PS) is separated from the reaction mixture in such a form that the recovery can be effected by simple filtration without need for precipitations by addition of solvents.
The main advantage of the process according to the invention is the possibility, unexpected in view of the prior art, to carry out the transphosphatidylation reaction of phosphatidylcholine and of similar phosphatides in an aqueous medium, to obtain phosphatidylserine of good purity and in highly satisfactory yields.
The features and the advantages of the process of the invention will be further disclosed by the following examples.